Calcium-conducting channels in the plasma membrane can appear very diverse (Parekh & Putney 2005) including voltage-gated ion channels (VOC's), receptor-operated ion channels (ROC's), but also store-operated channels (SOC's; Putney, 1986) that are activated in response to a decrease of the intraluminal Calcium concentration within i.e. the endoplasmic reticulum (ER). The latter have been demonstrated to serve as the main Calcium entry mechanisms in non-excitable cells.
Amongst the distinct SOCs, the CRAC current (ICRAC) is certainly characterized best and displays biophysical features such as high selectivity for Calcium ions, low conductance, and inward rectification (Hoth & Penner, 1992; Hoth & Penner, 1993; Parekh & Penner, 1997; Lepple-Wienhues & Cahalan, 1996; Kerschbaum & Cahalan, 1999). There's substantial evidence that the channels conducting CRAC predominantly rely on two proteins, Orail and Stiml (Roos et al., 2005; Feske et al., 2006; Peinelt et al., 2006). Orail constitutes the channel pore within the plasma membrane (Prakriya et al., 2006; Vig et al., 2006), whereas Stiml has been demonstrated to function as the sensor of the luminal Calcium concentration (Liou et al., 2005; Zhang et al., 2006).
In a physiological setting, ICRAC is activated in response to the engagement of cell-surface receptors that positively couple to phospholipase C (PLC). PLC increases the concentration of the soluble messenger inositol-1,4,5-trisphosphate (IP3), which opens ER membrane-resident IP3-receptors. Thus, IP3 triggers the release of Calcium from internal stores resulting in a drop of the luminal Calcium concentration (Lewis, 1999), which is sensed by Stiml. The Stiml molecule undergoes conformational changes inducing clustering with other Stiml molecules just underneath the plasma membrane. At these sites, Stiml can open the Orail pore by bridging the ER-PM gap with its C-terminal tail (Zhang et al., 2005; Luik et al., 2006; Soboloff et al. 2006, Wu et al. 2006; Li et al., 2007).
The above described process serves in signaling pathways of immune cells such as lymphocytes and mast cells. I.e. the activation of antigen or Fc receptors stimulates the release of Calcium from intracellular stores, and subsequent activation of ICRAC that impacts on downstream processes such as gene expression and cytokine release (Feske, 2007; Gwack et al., 2007; Oh-hora & Rao 2008).
The major contribution ICRAC provides to these signaling events has been convincingly demonstrated in patients suffering from severe combined immunodeficiency (SCID) due to a defect in T-cell activation. T cells and fibroblasts from these patients exhibited a strong attenuation of store-operated Calcium entry carried by ICRAC (Feske et al., 2006). This suggests CRAC channel modulators to serve as treatment in disease states caused by activated inflammatory cells.
The activation of antigen or Fc receptors stimulates the release of Calcium from intracellular stores and subsequent, sustained activation of ICRAC. Calcium carried by ICRAC activates calcineurin (CaN), which dephosphorylates the transcription factor NFAT. Upon dephosphorylation, NFAT shuttles into the nucleus and regulates gene expression in various ways depending on the nature of the stimulus as well as on the cell/tissue type.
NFAT participates in the transactivation of cytokine genes that regulate T-cell proliferation and other genes that control immune responses. Taking into account that the expression of cytokines such as IL-2, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor alpha (TNFα), granulocyte colony-stimulating factor (G-CSF), and gamma-interferon (INFγ) is prone to be controlled via transcriptional elements for NFAT, the impact of the ICRAC/CaN/NFAT signaling pathway on pro-inflammatory processes becomes apparent. The inhibition of this pathway has been demonstrated to be efficacious in patients by the use of drugs such as CsA and FK506, which act by inhibiting CaN.
A hallmark of ICRAC signaling in immune cells is that downstream processes such as gene expression rely on sustained Calcium entry rather than transient signals. However, Calcium entry is essential for other processes that can be independent of CaN/NFAT. Direct, Calcium-mediated release of substances (degranulation) such as histamine, heparin, and TNFα occur in i.e. mast cells, and are of rather acute nature. On the molecular level, this already points towards a differentiation potential for ICRAC blockers from calcineurin inhibitors.
Recent findings suggest that CRAC channel modulators can serve as treatment in disease states caused by the activation of inflammatory cells without side effects observed under treatments with i.e. steroids. Such diseases may include but are not limited to asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases such as multiple sclerosis, and disorders of the immune system.
U.S. Pat. No. 6,958,339, WO 2009/076454 A1, WO 2009/089305 A1, and WO 2010/122089 A1 each disclose a series of pyrazole carboxamide derivatives that are said to possess CRAC channel inhibitory activity which are believed to be useful in the treatment of allergic, inflammatory or autoimmune diseases.
Other small molecules possessing structurally different scaffolds as ICRAC inhibitors are known for instance from WO2005/009539, WO 2007/087427 A2 and WO 2007/087441 A2.
Pyrazole carboxamides as biologically active compounds are also known in the art, e.g. from EP 1176140 B1, US 2006/0100208 A1, WO 2005/016877 A2, WO 2006/076202 A1, WO 2007/002559 A1, WO 2007/024744 A2, WO 2009/011850 A2 and WO 2009/027393.